The field of biomolecule screening for biologically and therapeutically relevant compounds is rapidly growing. Relevant biomolecules that have been the focus of such screenings include chemical libraries, nucleic acid libraries, and peptide libraries in search for molecules that either inhibit or augment the biological activity of identified target molecules. With particular regard to peptide libraries, the isolation of peptide inhibitors of targets and the identification of formal binding partners of targets has been a key focus. However, one particular problem with peptide libraries is the difficulty of assessing whether any particular peptide has been expressed, and at what level, prior to determining whether the peptide has a biological effect.
The green fluorescent protein from Aequorea victoria (hereinafter “aGFP”) is a 238 amino acid protein displaying autofluorescent properties. The crystal structure of the protein and several point mutants has been solved (Ormo, M. et al. (1996) Science 273: 1392–95; Yang. F. et al. (1996) Nature Biotechnol. 14: 1246–51). The fluorophore, consisting of a modified tripeptide, is buried inside a relatively rigid β-can structure, where it is almost completely protected from solvent access. The protein fluorescence is sensitive to a number of point mutations (Phillips, G. N. (1997) Curr. Opin. Struct. Biol. 7: 821–27). Since any disruption of the structure allowing solvent access to the fluorophoric tripeptide results in fluorescence quenching, the fluorescence appears to be a sensitive indication of the preservation of the native structure of the protein.
Uses of GFP as a biological marker, such as gene expression, protein targeting, protein interactions, and biosensors, are well known. The extensively examined aGFP folds efficiently at or below room temperature, but fails to fold properly at higher temperatures. Aggregation of the protein appears to occur when overexpressed in certain organisms, resulting in weak fluorescence. In addition, the fluorescence of the native aGFP has a low quantum yield, which has prompted a search for variants of aGFP with improved stability and fluorescence properties. Although expression of aGFP is generally non-toxic to the cell in which it is expressed, there is some suggestion that aGFP is cytotoxic and may induce apoptosis in expressing cells (Liu, H. S. et al. (1999) Biochem. Biophys. Res. Commun. 260: 712–17). Finally, aGFP has been used as a scaffold for peptide display. However, some peptide insertions at the surface loops of aGFP result in low fluorescence, which suggests that aGFP may be sensitive to structural perturbations.
In view of the physical and biological properties of aGFP, other forms of GFPs are desirable with fluorescence and stability characteristics different from aGFP. Green fluorescent proteins have been cloned from Renilla reniformis (hereinafter “rrGFP”), Renilla muelleri (hereinafter “rmGFP”), and Ptilosarcus gurneyi (hereinafter “pGFP”) (see WO 99/49019, hereby expressly incorporated by reference). The core chromophore sequence of the rGFP and pGFPs is different from aGFP, and the Renilla forms have fluorescence characteristics with higher molar absorbance coefficient and narrower absorption/emission spectra as compared to aGFP (Ward, W. W. et al. (1979) J. Biol. Chem. 254: 781–88). The lack of significant homology to aGFP suggests that Renilla and Ptilosarcus forms provide important alternatives to the extensively exploited aGFP. Accordingly, it is the object of the present invention to provide compositions and methods comprising rGFP and pGFP.